This application is a national stage filing of PCT/JP99/01057, filed on Mar. 4, 1999, under 35 U.S.C. xc2xa7371. This application claims priority under 35 U.S.C. xc2xa7119 and 365 to application number 10/223897 filed in Japan on Jul. 8, 1998.
The present invention relates to a plant expressing soybean glycinin, which is useful as an ingredient in food, and to a propagation material of the plant, and the invention belongs to the fields of agriculture, food, and the like.
Soybean-extract protein (which has soybean globulin for its main ingredients), which is obtained by extracting proteins from soybeans and acid-precipitating the proteins (pH 4.5), is useful for preventing and treating arteriosclerosis and coronary heart diseases because it lowers the serum cholesterol level in human; particularly, it has the remarkable effection lowering a total cholesterol level, an LDL-cholesterol level, and a triglyceride level in patients with hypercholesterolemia (Mercer, N. J. H., Carroll, K. K., Giovannetti, P. M., Steinke, F. H. and Wolfe, B. M., Nutr. Rep. Int., 35, 279-287 (1987); T. Imura, M. Tanaka, T. Watanabe, S. Kudo, T. Uchida and T. Kanazawa, Ther. Res., 17, 2451-2456 (1996); M. Kanno, Food Industry, 39(18), 59-68 (1996)). In addition, the protein extract significantly reduces a total cholesterol level in healthy persons whose cholesterol levels are within normal limits (Kito, M., Moriyama, T., Kimura, Y. and Kambara, H., Biosci. Biotech. Biochem., 57, 354-355 (1993); Kambara, H., Nohara T., Kito, M., Ther. Res., 14, 3197-3204 (1993)).
By inhibiting the absorption of cholesterol and bile acid, soybean proteins play a pivotal role in lowering the cholesterol level (M. Kanno, Food Industry, 39 (18), 59-68 (1996)). Namely, the inhibition of cholesterol absorption results in an increase in cholesterol synthesis in the liver. However, the re-absorption of bile acid is inhibited at the same time and, as a result, cholesterol in the liver is oxidized into bile acid. This reduces the hepatic cholesterol concentration: and enhances the uptake of serum cholesterol into the liver. Thus, the serum cholesterol level is lowered (M. Kanno, Food Industry, 39(18), 59-68 (1996)).
It has been known that, an indigestible fraction obtained by intensive digestion of soybean protein with a protease markedly lowers the serum cholesterol level (M. Kanno, Food Industry, 39(18), 59-68 (1996)). The primary structure of the peptide present in the indigestible fraction still remains to be clarified (M. Kanno, Food Industry, 39(18), 59-68 (1996)). On the other hand, it has been reported that a peptide derived from A1aB1b subunit of glycinin, which is a major constituent of soybean globulins, has the bile acid-binding activity (Y. Makino, Food Industry, 39(24), 77-87 (1996)), and thus, the above-mentioned indigestible fraction has been believed to be derived from the bile acid-binding peptide originating from the A1aB1b subunit (M. Kanno, Food Industry, 39(18), 59-68 (1996)). Furthermore, a hydrophobic peptide derived from soybean protein is known to have bile acid-binding activity (Iwami, K., Sakakibara, K. and Ibuki, F., Agric. Biol. Chem., 50, 1217-1222 (1986)). Glycinin is the most hydrophobic protein among soybean globulins and there are two hydrophobic regions in the bile acid-binding peptide of A1aB1b subunit (S. Utsumi, Food Industry, 40(10), 68-79 (1997)). Consequently, it can be considered that, among soybean proteins, glycinin chiefly has the activity lowering the serum cholesterol level and, particularly, A1aB1b subunit is highly responsible for the activity.
Examples of industrial application of soybean-extract protein in the field of food include the following commercial products: Soybean Kara-age (fried soybean) and Protein Ganmo (fried tofu, where tofu is soybean curd) from Fuji Oil Co., Ltd.; G-9 and G-9 100 from Kanesa Co.; and Teriyaki Meatball (grilled meatball in teriyaki style), Hamburger, Pork Frankfurt from Nippon Meat Packers, Inc. However, there has been no report on functional food to which glycinin itself is added.
Furthermore, in the field of agriculture, there is no report on an attempt of giving the properties provided by soybean glycinin to other plants.
The present inventors focused on the above-mentioned properties of soybean glycinin and then predicted that the properties of soybean such as an activity of lowering the cholesterol level should be transplantable to plant species other than soybean by expressing soybean glycinin in these plants. The present inventors further focused on not only the above-mentioned activity of lowering the cholesterol level but also the facts that soybean glycinin protein is water soluble and has a wide variety of uses for processing food like tofu and that it excels nutritionally because it contains an essential amino acid xe2x80x9clysinexe2x80x9d abundantly, which is less abundant in rice. Thus, the inventors considered that it could be possible to produce new-type processed foods and new agricultural products having higher nutritive values by expressing soybean glycinin protein in other agricultural plants. Thus, an objective of the present invention is to provide a plant expressing soybean glycinin, particularly agricultural products useful as food.
The rice plant, of which seed, rice, is indispensable to Japanese eating habits, is one of the most important agricultural products. A major storage protein, glutelin, accounts for 80% of total protein in rice. Glutelin exhibits 32 to 37% homology to soybean glycinin at the amino acid sequence level, and the two proteins share a fundamental structure. Specifically, both glycinin and glutelin are synthesized as prepro-forms consisting of a signal peptide, A chain, and B chain, converted to pro-forms, and then processed to mature forms by being processed at specific sites of which sequences are highly conserved in each other. There are some differences between them; glycinin forms a trimer and then a hexamer by molecular association and is soluble in a salt solution; glutelin is insoluble in a salt solution because of its own giant molecule formed by disulfide bonding and hydrophobic interaction. Nonetheless, based on the similarity of their fundamental structures, the two proteins are considered to have evolved from the common ancestral gene (FIG. 1). Thus, just as with glutelin, it may be possible, by using glutelin gene promoter, to express and accumulate soybean glycinin, which is excellent in nutrition and processing suitability and has the effect on maintaining and promoting health by lowering the serum cholesterol level in human, in rice seeds, and then new values are thought to be added to rice while the wild-type characteristics of rice are maintained. Furthermore, the accumulation of a hybrid protein of glycinin and glutelin is also promising. Hence,the present inventors selected especially the rice plant as a plant to be used for the expression of soybean glycinin and attempted to express and accumulate soybean glycinin in rice, which is seed of rice plant and is one of useful agricultural products.
Specifically, a region containing the glutelin gene promoter, which expresses a gene specifically in the endosperm of rice plant seed, was isolated; a vector was constructed to have the natural or modified soybean glycinin gene ligated downstream of the promoter; the chimera gene construct was introduced into a cultured cell of rice plant; and thus, a transgenic rice plant was obtained. Subsequently, the inventors studied the tissue specificity and form of soybean glycinin expressed in the created transgenic rice plant. Then, they found that the expressed soybean glycinin was accumulated in rice plant seeds in the transgenic rice plant and the accumulated soybean glycinin was present as a matured protein formed by protein processing. Namely, the present inventors succeeded in expressing and accumulating soybean glycinin as a functional form in rice plant seeds and thus completed the present invention.
The present invention relates to a plant cell and a plant into which the soybean glycinin gene is introduced, preferably relates to a rice plant, and its propagation material, and more specifically relates to:
(1) a transformed plant cell expressively carrying a gene encoding a soybean glycinin protein;
(2) the transformed plant cell of (1), wherein the soybean glycinin protein is A1aB1b subunit;
(3) the transformed plant cell of (1) or (2), wherein the gene encoding a soybean glycinin protein is connected downstream of rice glutelin gene promoter;
(4) a transgenic plant comprising the transformed plant cell of any one of (1) to (3);
(5) the transgenic plant of (4), wherein the transgenic plant is a Poaceae plant;
(6) the transgenic plant of (4), wherein the transgenic plant is a rice plant;
(7) the transgenic plant of any one of (4) to (6), at least in a part of which soybean glycinin protein is accumulated;
(8) a propagation material of the plant of any one of (4) to (6);
(9) the propagation material of (8), wherein the propagation material is a rice seed; and
(10) the propagation material of (8) or (9), in which soybean glycinin protein is accumulated.
The present invention relates to a plant cell and a plant into which a gene encoding soybean glycinin protein is introduced and relates to its propagation material.
Soybean glycinin are composed of six subunits, in which A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3 subunits are assembled nearly at random. (For the structures and sequences of the subunits of glycinin, refer to: Utsumi, S. et al., Marcel Dekker, 257-291, 1997; Utsumi, S. et al., J. Agric. Food.Chem., 35, 210-214, 1987; Cho, T.-J. and Nielsen, N. C., Nucl. Acids Res., 17, 4338, 1989; Utsumi, S. et al., Agric. Biol. Chem., 51, 3267-3273, 1987; Fukazawa, C. et al., J. Biol. Chem., 260, 6234-6239, 1985; Momma, T. et al., Eur. J. Biochem., 149, 491-496, 1985.) In the present invention, there is no particular limitation on the genes encoding soybean glycinin protein to be expressed in a plant cell, as far as the genes encode the soybean glycinin subunits. However, particularly preferred is the gene encoding A1aB1b subunit (SEQ ID NO: 1), which has been known to show the effect on lowering the cholesterol level, etc., or the gene encoding A1bB2 or A2B1a subunit, which is thought to exhibit a similar effect. In the present invention, these subunits may be expressed singly or in combination in a plant cell.
Furthermore, a functional derivative of soybean glycinin protein may also be expressed instead of the natural one. The xe2x80x9cfunctional derivativexe2x80x9d means a protein of which amino acid sequence has one or more amino acid substitutions, deletions, and/or additions as compared with that of the natural protein and which is functionally equivalent to the natural protein. The term xe2x80x9cfunctionally equivalentxe2x80x9d means that the mutated protein has the activity of lowering the cholesterol level, characteristics for food processing, and/or nutritive properties equivalent to those of the natural protein. While the functional derivatives can be naturally occurring, they can also be prepared artificially. The artificial preparation method includes, for example, a method of substituting a particular amino acid(s) (one or more residues) with other amino acids, deleting a particular amino acid(s) (one or more residues), or inserting an amino acid(s) (one or more residues) at a particular site in a site-specific manner by using a single-stranded oligonucleotide, as well as a method of altering amino acid sequence by inserting, substituting, or deleting a double-stranded foreign gene or a synthetic gene using a particular restriction site (refer to Utsumi, S., Adv. Food Nutr. Res., 36, 89-208, 1992).
There is no particular limitation on the plant cell into which the soybean glycinin gene is to be introduced, and it is possible to use a cell from any plant species. However, particularly preferred is a cell of agricultural crops because the purpose of the present invention is to exert the function of soybean glycinin such as the function lowering the cholesterol level in the living body. The agricultural crops include, for example, grain crops such as rice plant, barley, wheat, rye, and maize; leguminous crops such as haricot, faba bean, and garden pea; oilseed crops such as peanut, sesame, colza, cotton, sunflower, and safflower; crops forming tuberous root such as potato and sweet potato; crops having fruit such as apple, melon, and grape; and crops of which leaf is used for food such as spinach, qing-geng-cai, and cabbage. In the present invention, the plant cell into which the soybean glycinin gene is to be introduced includes every type of plant cell capable of growing to be a plant. For example, this includes a cultured cell, protoplast, shoot primodium, multiple shoot, hairy root, and callus, but is not limited to these examples. The plant cell in the invention includes a cell in a plant.
In order to express the soybean glycinin gene in a plant cell, a DNA molecule comprising the following elements is introduced into the plant cell: (i) a promoter sequence having the activity of transcription activation in the plant cell, (ii) the soybean glycinin gene ligated downstream of the promoter sequence in sense-orientation, and (iii) terminator sequence, which contains a sequence segment required for transcription termination and polyadenylation, ligated downstream of the gene. Such a DNA molecule may contain a DNA sequence element that is capable of enhancing the transcription, other than the promoter element, for example, an enhancer sequence.
There is no particular limitation on the promoter to be used, as far as the promoter is functional in the plant cell. However, preferred is a tissue-specific promoter that secures the effective expression of the glycinin gene at a desired site in a regenerated plant. Preferable tissue-specific promoters include, for example, glutelin gene promoter for the expression in rice plant seed (Takaiwa, F. et al., Plant Mol. Biol., 17, 875-885, 1991); glycinin gene promoter or a promoter of the major storage protein gene in each crop for the expression in the seeds of leguminous crops such as haricot, faba bean, and garden pea or oilseed such as peanut, sesame seed, colza, cotton seed, sunflower, and safflower; for example, phaseolin gene promoter for the expression in haricot (Murai, N. et al., Science, 222, 476-482, 1983), and cruciferin gene promoter for the expression in colza (Rodin, J. et al., Plant Mol. Biol., 20, 559-563, 1992); furthermore, patatin gene promoter for the expression in the tuber of potato (Rocha-Sosa, M. et al., EMBO J., 8, 23-29, 1989), and sporamin gene promoter for the expression in the tuberous root of sweet potato (Hattori, T. and Nakamura, K., Plant Mol. Biol., 11, 417-426, 1988); ribulose-1,5-bisphosphate decarboxylase gene promoter in a leaf of vegetable such as spinach (Orozco, B. M. and Ogren, W. L., Plant Mol. Biol., 23, 1129-1138, 1993). These specific examples of promoter are just illustrative, and a variety of promoters other than these are usable for each practical purpose. In addition, it is possible to use not only the above-mentioned tissue-specific promoters but also constitutive promoters such as 35S promoter as well as inducible promoters.
Introduction of the soybean glycinin gene into a plant cell can be achieved by various methods that are known to those skilled in the art. For example, the following method can be used: an indirect introduction method using Agrobacterium tumefaciens or Agrobacterium rhizogenes (Hiei, Y. et al., Plant J., 6, 271-282, 1994; Takaiwa, F. et al., Plant Sci. 111, 39-49, 1995) or a direct introduction method represented by electroporation method (Tada, Y. et al. Theor. Appl. Genet, 80, 475, 1990), polyethylene glycol method (Datta, S. K. et al., Plant Mol Biol., 20, 619-629, 1992), or particle gun method (Christou, P. et al., Plant J. 2, 275-281, 1992; Fromm, M. E., Bio/Technology, 8, 833-839, 1990).
Regeneration of the transformed plant cell enables creating a plant. The method of the regeneration depends on the types of plant cells; representative methods are, for example, the method of Fujimura et al. for rice plant (Fujimura, T. et al., Plant Tissue Culture Lett., 2, 74, 1995); the method of Armstrong et al. for maize (Armstrong, C. L. and Phillips, R. L., Crop Sci., 28, 363-369, 1988); the method of Radke et al. for colza (Radke S. E., Theor. Appl. Genet. 75, 685-694, 1988); and the method of Sheerman et al. for potato (Sheerman, S. and Bevan, M. W., Plant Cell Rep., 7, 13-16, 1988).
Because of the expression of introduced soybean glycinin gene, soybean glycinin protein is expressed and accumulated in target regions of a plant originating from the created transgenic plant or its propagation material, for example, seed, tuberous root, tuber, fruit, cutting, etc. Thus, the characteristics of soybean glycinin protein, such as nutritive properties, characteristics for food processing, and promoting activity for health, can be imparted to other plant species.